Recently, development of basic sciences, such as developmental biological engineering and stem cell biology, and advances in recombinant vector technologies for gene therapy, etc. have brought about a rapidly growing interest in regenerative medicine. Now, ex vivo and in vivo proliferation and tissue formation technologies are investigated for wide varieties of functional cells and organs including skin, cartilage, bone, blood, blood vessels, nerve, cardiac muscle, pancreatic islets and liver. Details of these investigations are described, for example, in Regenerative Medicine and Life Science, Extra Issue to September 2000 Issue of Protein, Nucleic Acid and Enzyme (Kyoritsu Shuppan, Co.); Regenerative Medicine, Special Issue of The Medical Frontline (Saishin Igaku Co.); and Molecular Medicine, Vol. 38 No. 1 (Nakayama Shoten).
One of the common problems that regenerative medicine must overcome is the supply of cell sources. The cell sources should be determined by whether they possess the cellular functions required for each target cell, tissue or organ and whether sufficient number of cells can be obtained, i.e., both properties of function and proliferation of the cells are required. At the current technical level, cell sources satisfying these requirements are not obtained sufficiently, except for simple tissues such as skin. Recently, however, induction of proliferation/differentiation of embryonic stem cells (ES cells), induction of proliferation/differentiation of adult stem cells (including those from newborns), and methods for temporarily immortalizing or dedifferentiating terminally differentiated functional cells have attracted attention. ES cells are thought to be able to differentiate totipotently into all cell types/tissues since ES cells can generate individuals. It has been reported that ES cells actually differentiate into diverse cell types in mouse (see the Chapter written by Niwa in Regenerative Medicine and Life Science, supra). Since human ES cells have been established recently, application of these cells to medical treatment is expected (see Chapter written by Nakatsuji in Regenerative Medicine and Life Science, supra). On the other hand, the presence of various types of stem cells, such as hematopoietic stem cells, neural stem cells and bone marrow mesenchymal stem cells, has been confirmed even in the adult body (see the Chapters written by Nakauchi, Okano and Fukuda in Regenerative Medicine and Life Science, supra). Of these, those stem cells other than hematopoietic stem cells are attracting attention as cell sources for, in particular, autologous cell transplantation since ex vivo proliferation technology is applicable to them. Neuronal stem cells are also attracting attention as a cell source for the same purpose since they are capable of differentiating into all germ layers (Clarke et al., Science 288:1660). With respect to immortalization of terminally differentiated functional cells, it is reported that human hepatocytes can be rendered proliferative property by transdution of an immortalizing gene and that the cells restore their innate functions as hepatocytes by deletion of the immortalizing gene that may cause cancer (Kobayashi et al., Science 287:1258).
In order to improve the proliferation of these cell sources, to induce differentiation into cells of a specific lineage, to control differentiation/dedifferentiation, and to control immortalization/deimmortalization, they have been studied that foreign genes are transduced into cells and that their expressions are controlled. There are known methods for controlling the expression of foreign genes transduced into cells, such as—methods using conditional expression control systems, methods using site-specific recombinases, etc.
The following conditional expression control systems for mammal cells are known: 1) systems using a thermo-sensitive promoter or a thermo-sensitive mutant of a foreign gene; 2) systems using a heavy metal ion-inducible promoter; 3) systems using a hormone-inducible promoter; 4) systems using a hypooxic-inducible promoter; 5) systems using a cytokine-inducible promoter; 6) control systems inducible by tetracycline; 7) control systems inducible by FK506 (also known as rapamycin); 8) control systems inducible by RU486 (also known as mifepristone); 9) control systems inducible by ecdysone; and 10) control systems inducible by streptogramin. The conditional expression control systems described in 1) to 5) above are classic systems, and are not appropriate for controlling gene expression in mammalian cells, because their basal expression levels are rather high and they produce diverse effects on cells other than the control of a target gene.
On the other hand, the conditional expression control systems described in 6) to 10) above were developed recently in order to overcome these problems. For 6), see Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, 17.52–17.59 and Gossen et al. (Proc. Natl. Acad. Sci. USA, 89:5547–5551 and Science 268:1766–1769); for 7), see Magari et al. (J. Clin. Invest. 100:2865–2872) and Clackson (Current Opinion in Chemical Biology 1:210–218); for 8), see Harvey et al. (Current Opinion in Chemical Biology 2:512–518); for 9), see Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, 17.71–17.74 Saez et al. (Proc. Natl. Acad. Sci. USA 87:14512–14517) and No et al. (Proc. Natl. Acad. Sci. USA, 93:3346–3351); and for 10), see Fussenegger et al. (Nature Biotechnology, 18:1203–1208).
Among these, control systems inducible by tetracycline are most commonly used. This type of system was developed by Gossen et al. (Proc. Natl. Acad. Sci. USA, 89:5547–5551 and Science 268:1766–1769). This system, being induced by tetracycline or its derivatives such as doxycycline (hereinafter, collectively referred to as “Tet”), can start (Tet-on) or terminate (Tet-off) the expression of any gene of interest. This mechanism can be explained as follows, taking the Tet-off control system as an example. Briefly, Tet transactivator (tTA) protein forms a complex with an antibiotic Tet (a low molecular weight compound). This complex binds to tandem sequences of Tet-response element (TetO, a 19-mer DNA sequence) to thereby suppress the activity of a minimal promoter located in the vicinity, suppressing the expression of a target gene of interest linked to downstream of the promoter. In the present specification, the combination of the TetO tandem sequences and the minimal promoter is simply referred to as “Tet responsive promoter”.
In control systems inducible by tetracycline, an expression system for tTA protein is required in addition to the Tet responsive promoter. Usually, a gene encoding tTA protein is ligated downstream of a constitutive promoter in a separate vector in order to express this protein. The tTA protein is a fusion protein composed of an E. coli transposon 10 (Tn10)-derived Tet repressor (TetR) protein that binds to TetO in the absence of Tet and a herpes simplex virus (HSV)-derived VP16 having transcription activating effect. On the other hand, the Tet-on control system employs, instead of the tTA protein, its mutant protein called reverse Tet transactivator (rtTA) protein having 4 amino-acid mutations in the tTA protein. This rtTA protein suppresses the transcriptional activity of the Tet responsive promoter in the absence of Tet, and activates the same in the presence of Tet.
Various improvements have been made to tetracycline-inducible control systems. These improvements include the preparation of a single vector comprising both a control system for a gene of interest and an (r)tTA expression system (Paulus et al., J. Virol. 70:62–67); the preparation of mutants of (r)tTA and TetO, as well as a system switching between the expression of two genes depending on Tet concentration (Baron et al., Proc. Natl. Acad. Sci. USA, 96:1013–1018); a system that is allowed to have more stringent responsiveness to Tet concentration by a combination of Tet repressor and Tet activator (Rossi et al., Molecular Cell, 6:723–728); and a system that is made autoregulatory by expressing under the control of TetO promoter a reporter gene and a DNA encoding tTA bicistronically through an IRES (internal ribosome entry site from encephalomyocarditis virus) placed between the two genes, wherein basal expression levels have been reduced (Hofmann et al., Proc. Natl. Acad. Sci. USA, 93:5185–5190).
In expression control systems inducible by low molecular weight compounds other than Tet, the control of expression is performed by a mechanism almost the same as described above (Clackson, Current Opinion in Chemical Biology, 1:210–218). Expression control by similar mechanisms is also reported in the references cited above. The term “low molecular weight compound” used herein refers to a low molecular weight organic compound, including tetracycline, FK505, RU486, ecdysone, streptogramin and derivatives thereof.
As site-specific recombinases, several recombinases including Cre recombinase (Gorman et al., Current Opinion in Biotechnology 11:455–460) and FLP recombinase (Buchholz et al., Nature Biotechnology 16:657–662) have been found. These recombinases belong to the integrase family, and are reported to act in cells or individuals of higher organisms though they are derived from microorganisms (Sauer et al., Current Opinion in Biotechnology 5:521–527). Metzger et al. (Current Opinion in Biotechnology 10:470–476) reported creation of a transgenic mouse utilizing Cre recombinase; briefly, in order to turn on or turn off the expression of a foreign gene in a specific organ of a transgenic mouse or at any desired time, a first vector in which an organ-specific promoter or inducible promoter is ligated upstream of Cre recombinase gene and a second vector in which a foreign gene or a stuffer sequence preventing the expression of this gene is flanked by two loxP sequences were constructed and transferred into ES cells for creation of a transgenic mouse. FLP recombinase and its recognition sequence FRT are also examined in a similar manner as Cre/loxP are examined (see Westerman et al. cited below).
Westerman et al. constructed a retrovirus vector in which a simian virus-derived immortalizing gene, SV40 large T antigen gene, is flanked by two recognition sites of a site-specific recombinase, and transfected this vector into normal cells. As a result, improved proliferation and life span extension were observed in the cells. They report that by transfecting another retrovirus vector capable of expressing Cre recombinase into these cells, SV40 large T antigen gene can be excised (Proc. Natl. Acad. Sci. USA 93:8971–8976). Japanese Unexamined Patent Publication (Kohyo) No. 11-507230 (corresponding to WO96/40877 in which Anderson, David J. is named as an inventor) discloses a cell that has in its genome a structure in which an immortalizing gene is flanked by two target sites of a recombinase, the immortalizing gene being capable of excision by the recombinase. The method described in Japanese Unexamined Patent Publication (Kohyo) No. 11-507230 is a method of gene transfer using the vectors described in FIGS. 3C, 6A and 6B thereof. This method uses a vector in which a recombinase gene is placed downstream of an inducible promoter and, at the same time, an immortalizing gene flanked by two target sites of the recombinase is also placed (see FIG. 1—1 of the present specification). However, a combination of an inducible promoter and a recombinase gene falls within the classic expression control systems described 1) to 5) above, and allows high levels of basal expression (Sasz et al., Current Opinion in Biotechnology, 8:608–616). Even the control systems inducible by a low molecular weight compound described in 6) to 10) do not exert tight control over basal expression. Thus, their drawback is that it is difficult to suspend the expression of recombinase when its expression is not desired.
Taking the tetracycline-inducible control system as an example, the average ratio of [expression level in “on” mode]/[expression level in “off” mode] is only about 100-fold in the Tet-off control system, and only about 10-fold in the Tet-on control system according to Werner Paulus et al. (J. Biotechnology, 81:159–165). Therefore, it is difficult to apply these control systems to such a gene whose small amount of basal expression would give an irreversible effect on cells. Specific examples of such a gene include genes causing cytotoxicity, apoptosis-inducing genes, cell differentiation-inducing genes, immortalizing genes, oncogenes, and genes encoding proteins such as site-specific recombinases that cause recombination of nucleic acid sequences. St-Onge et al. (Nucleic Acids Research, 24:3875–3877) actually placed Cre recombinase under control of a tetracycline-inducible control system and examined its action. As a result, they report that leakage of Cre recombinase expression is observed at the basal level. Such leakage of expression at the basal level is the weakest point in low molecular weight compound-inducible control systems, and various researches are made toward its solution (Molecular Cloning, Third Edition, Cold Spring Harbor Laboratory Press, p. 17.56). Pollock et al. constructed a retrovirus vector in which a secreted alkaline phosphatase gene is placed under an inducible promoter so that a transcript of this gene from this promoter is sense or antisense to a transcript from the retrovirus vector LTR and an internal promoter; they report that little basal expression was observed in the antisense configuration (Proc. Natl. Acad. Sci. USA, 97:13221–13226).